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The quest for safe and effective ablation resulted in the development of nanosecond pulsed electric fields (nsPEF) technology for tumor treatment. For future applications of nsPEF in urothelial cancer treatment, we evaluated the effect of urine presence at the ablation site. We prepared artificial urine (AU) with compounds commonly present in the healthy human urine at physiological concentrations. We compared nsPEF cytotoxicity for cancerous (T24) and non-cancerous (SV-HUC-1) human urothelial cell lines pulsed either in the AU or in a physiological solution (PS). Cell monolayers were exposed to trains of 300-ns, 10-Hz pulses using a two-needle electrode assembly placed orthogonal to the monolayer. The assembly produced the electric field gradually weakening with the distance from the electrodes. The electric field which killed 50 % of cells (LD50) was measured by staining with propidium iodide and matching the stained area with the simulated electric field strength. nsPEF exposure in PS was more cytotoxic to cancer cells. The AU protected both healthy and cancer urothelial cells, increasing their LD50 1.4 and 1.6 times, respectively. Omitting urea from the AU reduced the LD50 for healthy and cancer urothelial cells. Testing the role of other AU components, we found that it was the high concentration of phosphates what also rendered the protective effect of the AU. Our findings suggest that the nsPEF ablation of bladder cancer will be less efficient if the bladder is filled with urine.

Neuromodulation applications of nanosecond electric pulses (nsEP) are hindered by their low potency to elicit action potentials in neurons. Excitation by a single nsEP requires a strong electric field which injures neurons by electroporation. We bypassed the high electric field requirement by replacing single nsEP stimuli with high-frequency brief nsEP bursts. In hippocampal neurons, excitation thresholds progressively decreased at nsEP frequencies above 20-200 kHz, with up to 20-30-fold reduction at sub-MHz and MHz rates. For a fixed burst duration, thresholds were determined by the duty cycle, irrespective of the specific nsEP duration, rate, or number of pulses per burst. For 100-μs bursts of 100-, 400-, or 800-ns pulses, the threshold decreased as a power function when the duty cycle exceeded 3-5 %. nsEP bursts were compared with single "long" pulses whose duration and amplitude matched the duration and the time-average amplitude of the burst. Such pulses deliver the same electric charge as bursts, within the same time interval. High-frequency nsEP bursts excited neurons at the time-average electric field 2-3 times below the threshold for a single long pulse. For example, the excitation threshold of 139 ± 14 V/cm for a single 100-μs pulse decreased to 57 ± 8 V/cm for a 100-μs burst of 100-ns, 0.25-MHz pulses (p 0.001). Applying nsEP in bursts reduced or prevented the loss of excitability in multiple stimulation attempts. Stimulation by high-frequency nsEP bursts is a powerful novel approach to excite neurons at paradoxically low electric charge while also avoiding the electroporative membrane damage.

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca2+]. We explored the impact of Ca2+ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca2+ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca2+-free medium, perfusion with 2 mM of external Ca2+ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca2+-independent resealing (τ = 13–15 s) and slow, Ca2+-dependent processes (τ ~70 s with Ca2+ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca2+ (by loading cells with BAPTA or after Ca2+ depletion from the reticulum), suggesting the recruitment of unknown Ca2+-independent repair mechanisms.

Stimulation and electroporation by nanosecond electric pulses (nsEP) are distinguished by a phenomenon of bipolar cancellation, which stands for a reduced efficiency of bipolar pulses compared to unipolar ones. When two pairs of stimulating electrodes are arrayed in a quadrupole, bipolar cancellation inhibits nsEP effects near the electrodes, where the electric field is the strongest. Two properly shaped and synchronized bipolar nsEP overlay into a unipolar pulse towards the center of the electrode array, thus canceling the bipolar cancellation (a "CANCAN effect"). High efficiency of the re-created unipolar nsEP outweighs the weakening of the electric field with distance and focuses nsEP effects to the center. In monolayers of CHO, BPAE, and HEK cells, CANCAN effect achieved by the interference of two bipolar nsEP enhanced electroporation up to tenfold, with a peak at the quadrupole center. Introducing a time interval between bipolar nsEP prevented the formation of a unipolar pulse and eliminated the CANCAN effect. Strong electroporation by CANCAN stimuli killed cells over the entire area encompassed by the electrodes, whereas the time-separated pulses caused ablation only in the strongest electric field near the electrodes. The CANCAN approach is promising for uniform tumor ablation and stimulation targeting away from electrodes.